The long-term aim of this project is to understand the structural components of BK-type Ca2+- and voltage- activated K+ channels and their functional properties and physiological roles. BK channels are widely expressed and couple changes in submembrane Ca2+ concentrations to changes in membrane potential and excitability. How they do this varies in a tissue-specific fashion dependent in part on molecular composition. They consist minimally of four pore-forming 1 subunits. In addition, there are four distinct genes that encode auxiliary 2 subunits. Two of the 2 subunit family members, 22 and splice variants of 23, produce rapid inactivation in which the cytosolic N-terminus of the 2 subunits moves into a blocking position within the BK channel pore. However, in contrast to rapid inactivation of voltage-dependent K+ channels, inactivation of BK channels occurs by a two-step mechanism, in which a fully-conducting, pre-inactivated open state precedes the inactivated state. An implication of this mechanism is that it enables BK 2 subunits to regulate afterhyperpolarizations following development of inactivation. In fact, the 23a subunit results in a profound increase in the net current flux through BK channels following repolarization. This effect represents an entirely new mechanism by which slow use-dependent afterhyperpolarizations can be generated and will have profound effects on excitability of cells in which it is found. This project will be pursued in two parts. In part one, two aims are devoted to understanding the mechanism and structural basis of inactivation mediated by these 22 and 23 subunits. In part two, the potential physiological consequences of this inactivation mechanism and the tissue specific localization of the key 2 subunits will be determined. For the mechanistic studies and examination of physiological consequences, methods of electrophysiology combined with molecular biology will be employed. Methods of PCR and immunohistochemistry will be used to identify loci of expression of 22 and 23 subunits. Together these results will provide insight into a new type of regulatory mechanism that may have profound significance for regulation of excitability in cells containing BK 2 subunit. BK channels are of broad importance in the normal functioning of a variety of excitable cells. Among different tissues, BK channels contribute to regulation of neuronal excitability, smooth muscle relaxation, synaptic transmission and hormone release. Better understanding the composition and functional role of BK channel variants is of potential medical importance, not only because the channels may serve as specific therapeutic targets but also because altered function of particular variants may contribute to currently unrecognized pathological conditions. Calcium and voltage-regulated potassium channels (BK-type) are widely distributed among a range of cell types and contribute to regulation of neuronal excitability, smooth muscle relaxation, synaptic transmission and hormone release. These channels have been implicated in pathological conditions as diverse as hypertension and epilepsy. Better understanding the composition and functional role of BK channel variants is of potential medical importance, not only because the channels may serve as specific therapeutic targets but also because altered function of particular BK variants may contribute to currently unrecognized pathological conditions.